Pyrosequencing Analysis of APC Methylation Level in Human Prostate Tissues: A Molecular Marker for Prostate Cancer

DNA hypermethylation-induced silencing of tumor suppressor and DNA repair genes is a frequent phenomenon in cancer [1-4]. These hypermethylation markers are promising tools for detecting cancer cells in tissues and body fluids, and various methods for detecting aberrant DNA methylation have been developed in recent years. Which of these analytical methods is best suited for use in the clinical setting, however, remains unclear [2-6]. The majority of previous studies used methylation-specific polymerase chain reaction (MSP) or quantitative methylation-specific polymerase chain reaction (QMSP) to assess methylation and often artificially classified the data into simplistic hypermethylated or hypomethylated categories [2-4]. Despite common use, these methods have known disadvantages, including the generation of false-positive and false-negative results [7, 8]. Pyrosequencing (PSQ) is one of the most accurate methods available for quantifying DNA methylation [3, 7, 8]. It is a sensitive and highly reproducible method that is uniquely suited to the analysis of clinical specimens from which only small amounts of DNA can be isolated [7, 8]. To the best of our knowledge, relatively few studies of methylation in PCa have used the PSQ method [8, 9].

Adenomatous polyposis coli (APC) is a well-characterized tumor suppressor gene that regulates the Wingless-type (Wnt) signaling pathway via ubiquitin-mediated beta-catenin degradation. APC is consistently hypermethylated during PCa development, and APC hypermethylation has well-established diagnostic and prognostic significance [10-16]. The aim of the present study was to evaluate the relevance of APC methylation in discriminating between benign prostatic hyperplasia (BPH) and PCa and to determine whether APC methylation is associated with clinicopathological parameters by use of quantitative PSQ analysis.

Our results showed that APC methylation levels are significantly higher in PCa patients than in BPH patients. Moreover, APC hypermethylation was not only associated with the increased incidence of PCa but was also positively correlated with increased Gleason score. By contrast, no significant relation was detected between APC methylation and age, stage, or PSA level in PCa patients.

Epigenetic alterations, such as abnormal DNA-methylation patterns, are associated with many human tumor types [2-4]. Differences in methylation patterns have also emerged as markers for cancer risk assessment, cancer diagnosis, and therapy monitoring in several different types of cancer [2-4]. DNA methylation is quite useful in cancer detection owing to the inherent stability of DNA compared with RNA or proteins [4]. As a disease marker, methylation is useful regardless of whether it functions in gene silencing or not, as long as it is specific to tumor cells or is associated with clinically important information [3, 4]. In this regard, the present results suggest that the detection of APC methylation by PSQ has promising diagnostic value in PCa owing to its high sensitivity (89.3%) and specificity (98.1%).

Recently, several methodologies have become available to detect the methylation status of certain genes in clinical samples [3, 4]. However, the results depend on the detection method, including the primer design, reagents, detectors, equipment, and protocols, which influences sensitivity and specificity. The use of conventional MSP is limited in cancer detection because benign lesions can be weakly positive and cannot be distinguished from cancer cases. Moreover, the results of MSP in any particular DNA region are reported simply and perhaps subjectively as methylated or unmethylated, without allowing the quantitation or identification of partial methylation. Quantitative analysis of DNA methylation status with appropriate methods might improve the accuracy of interpretation obtained from small amounts of DNA in clinical samples. In that respect, PSQ might be a better method because it provides quantitative information for each target CG site instead of qualitative information. In the present study, PSQ was used to measure methylation, and an optimal threshold level was identified to discriminate between PCa and normal controls. The threshold was relatively low (6.07%) and might therefore be misleading with other techniques [3, 7, 8]. One of the major advantages of PSQ is the ability to compare samples quantitatively and to segregate various pathologic covariates accurately on the basis of methylation levels. Without PSQ, no significant correlation between APC methylation level and Gleason score could be detected in the present study.

APC is a well-characterized tumor suppressor. It down-regulates Wnt signaling by targeting the transcriptional coactivator beta-catenin for proteasomal degradation, thereby preventing its association with the nuclear transcription factor T-cell factor/lymphoid enhancer factor [10]. The Wnt pathway plays a central role in tumorigenesis. Its inappropriate activation is a common feature of many human cancers, leading to the deregulation of cell proliferation and differentiation [17]. Initially identified in colorectal cancer, APC is inactivated in various malignancies, including PCa, by genetic and epigenetic mechanisms [11-16]. The methylation status of APC can be used to distinguish benign tissues from PCa [13-16]. Furthermore, the methylation status of APC correlates significantly with clinicopathological variables, including tumor stage, grade, and poor prognosis [11-14]. Our findings are consistent with previous results and indicate that APC hypermethylation is a reliable predictor of PCa and of its aggressive features [13, 14].

Although histologically confirmed prostate tissues were used, the possibility of unrevealed PCa in BPH patients and an undetected small fraction of methylated DNA might affect our results for sensitivity and specificity. Additionally, determination of the Gleason score with different surgical methods such as TUR and prostatectomy may be a limitation of our study. Nonetheless, promising methylation frequency results were obtained. The sensitivity of APC methylation analysis by PSQ and its specificity for PCa over benign tissue reached 89.3% and 98.1%, respectively. Moreover, the frequency of APC methylation in PCa was independent of serum PSA level, Gleason score, and stage. Although these findings are promising, these kinds of studies must be performed with body fluids (urine and blood) to have clinical relevance. For these reasons, multicenter, large-scale clinical validation studies using primary human cancer tissues and body fluids are currently underway at our institute to confirm APC as a diagnostic methylation marker for PCa. These studies will improve our understanding of the biological role and clinical relevance of APC methylation in tumorigenesis.

Our study demonstrates that APC methylation is associated with PCa and its aggressive tumor features.